Cystic fibrosis (CF) is a genetic disease, causing thick mucus to buildup in the airways that is associated with chronic inflammation and persistent lung infection, ultimately leading to respiratory failure. Non- tuberculosis mycobacterial (NTM) infections, such as Mycobacterium abscessus (Mab), are among the most severe chronic infections associated with CF. Prolonged treatment with potent antibiotics is often met with low compliance due to the severe side effects, complicated by frequent recurrence and drug-resistant strains. SciBac has developed a novel live biotherapeutic using a proprietary microbial improvement platform called Directed Recombination by In Vitro Evolution (DRIVE), that transfers therapeutic traits to commensal microbial strains. This proposal focuses on the use of DRIVE technology to combine mucolytic and antibacterial traits to optimize a lactobacillus hybrid to demonstrate the safe and effective use in treating chronic Mab lung infection in a relevant mouse model. The hypothesis is that the DRIVE platform will generate hybrid strains that produce bacteriocins and metabolize mucin resulting in a stand-alone live biotherapeutic to treat chronic NTM lung infection. To this end we aim to first, optimize the efficacy of a Lactobacillus delbrueckii hybrid using DRIVE to transform NTM antibacterial activity from Bacillus subtilis. The ability of probiotic strains to survive in a competition rich environment is essential for the sustained delivery of therapeutic metabolites. The optimize L. delbrueckii hybrid will be characterized and new activities validated via time-kill analysis and qualitative mucin degradation assays in vitro. Then, safety and stability of optimized L. delbrueckii hybrids will be validated in a cytotoxicity assay with human lung epithelial cells and cytokine production using primary human bronchial/tracheal epithelial cells (normal and CF) evaluated via ELISA. The genetic transfer will be fully characterized through comparative whole genome sequencing of donor, host, and hybrid strains and repeated passage. Preclinical safety in heathy mice and efficacy studies in a statistically relevant agar bead mouse model of chronic Mab infection will be used to demonstrate proof-of concept. Following treatment, both infected and uninfected mice lungs and spleens will be assessed for viable colony forming units (NTM and/or hybrid) and cytokine analysis, total cell count, cell differential analysis of the bronchoalveolar lavage fluid will also be completed. The results will determine initial safety and efficacy in animal models and provide support for future preclinical testing. The goal of this Phase I effort is to demonstrate proof-of-concept that live biotherapeutics are safe and effective in treating Mab lung infection.